Genetic Diversity of <Emphasis Type="Italic">Acacia seyal</Emphasis> Del. Rhizobial Populations Indigenous to Senegalese Soils in Relation to Salinity and pH of the Sampling Sites

The occurrence and the distribution of rhizobial populations naturally associated to Acacia seyal Del. were characterized in 42 soils from Senegal. The diversity of rhizobial genotypes, as characterized by polymerase chain reaction restriction fragment length polymorphism (RFLP) analysis of 16S–23S rDNA, performed on DNA extracted from 138 nodules resulted in 15 clusters. Results indicated the widespread occurrence of compatible rhizobia associated to A. seyal in various ecogeographic areas. However, the clustering of rhizobial populations based on intergenic spacer (IGS) RFLP profiles did not reflect their geographic origin. Four genera were discriminated on the basis of 16S rRNA gene sequences of the strains representative for the IGS-RFLP profiles. The majority of rhizobia associated to A. seyal were affiliated to Mesorhizobium and Sinorhizobium 64 and 29%, respectively, of the different IGS-RFLP profiles. Our results demonstrate the coexistence inside the nodule of plant-pathogenic non-N₂-fixing Agrobacterium and Burkholderia strains, which induced the formation of ineffective nodules, with symbiotic rhizobia. Nodulation was recorded in saline soils and/or at low pH values or in alkaline soils, suggesting adaptability of natural rhizobial populations to major ecological environmental stress and their ability to establish symbiotic associations within these soil environments. These results contribute to the progressing research efforts to uncover the biodiversity of rhizobia and to improve nitrogen fixation in agroforestry systems in sub-Saharan Africa.     

Different species and symbiotic genotypes of field rhizobia can nodulate Phaseolus vulgaris in Tunisian soils

A collection of 160 isolates of rhizobia nodulating Phaseolus vulgaris in three geographical regions in Tunisia was characterized by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-amplified 16S rDNA, nifH and nodC genes. Nine groups of rhizobia were delineated: Rhizobium gallicum biovar (bv.) gallicum, Rhizobium leguminosarum bv. phaseoli and bv. viciae, Rhizobium etli bv. phaseoli, Rhizobium giardinii bv. giardinii, and four groups related to species of the genus Sinorhizobium, Sinorhizobium meliloti, Sinorhizobium medicae and Sinorhizobium fredii. The most abundant rhizobial species were R. gallicum, R. etli, and R. leguminosarum encompassing 29–20% of the isolates each. Among the isolates assigned to R. leguminosarum, two-thirds were ineffective in nitrogen fixation with P. vulgaris and harbored a symbiotic gene typical of the biovar viciae. The S. fredii-like isolates did not nodulate soybean plants but formed numerous effective nodules on P. vulgaris. Comparison of nodC gene sequences showed that their symbiotic genotype was not related to that of S. fredii, but to that of the S. fredii-like reference strain GR-06, which was isolated from a bean plant grown in a Spanish soil. An additional genotype including 16% of isolates was found to be closely related to species of the genus Agrobacterium. However, when re-examined, these isolates did not nodulate their original host.     

A study of the impacts of Zn and Cu on two rhizobial species in soils of a long-term field experiment

Two agriculturally important species of rhizobia, Rhizobium leguminosarum biovar viciae (pea rhizobia) and R. leguminosarum bv. trifolii (white clover rhizobia), were enumerated in soils of a long-term field experiment to which sewage sludges contaminated predominantly with Zn or Cu, or Zn plus Cu, were added in the past. In addition to total soil Zn and Cu concentrations, soil pore water soluble Zn and free Zn²⁺, and soluble Cu concentrations are reported. Pea and white clover rhizobia were greatly reduced in soils containing ≥200 mg Zn kg^-1, and soil pore water soluble Zn and free Zn²⁺ concentrations ≥7 and ≥3 mg l^-1, respectively, in soils of pH 5.9–6. Copper also reduced rhizobial numbers, but only at high total soil concentrations (>250 mg kg^-1) and not to the same extent as Zn. Yields of field grown peas decreased significantly as total soil Zn, soil pore water soluble Zn and free Zn⁺² increased (R² = 0.79, 0.75 and 0.75, respectively; P < 0.001). A 50% reduction in seed yield occurred at a total soil Zn concentration of about 290 mg kg^-1, in soils of pH 5.9–6. The corresponding soil pore water soluble Zn and free Zn²⁺ concentrations were about 9 and 4 mg l^-1, respectively. Pea seed yields were not significantly correlated with total soil Cu (R² = 0.33) or soil pore water soluble Cu (R² = 0.39). Yield reductions were due to a combination of greatly reduced numbers of free-living rhizobia in the soil due to Zn toxicity, thus indirectly affecting N₂-fixation, and Zn phytotoxicity. These effects were exacerbated in slightly acidic soils due to increased solubility of Zn, and to some extent Cu, and an increase in the free Zn²⁺ fraction in soil pore water. The current United Kingdom, German and United States limits for Zn and Cu in soils are discussed in view of the current study. None of these limits are based on toxicity thresholds in soil pore water, which may have wider validity for different soil types and at different pH values than total soil concentrations. This revised version was published online in June 2006 with corrections to the Cover Date.     

The effect of biotic and physical factors on the competitive ability of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis>

Background  

Rhizobium leguminosarum bv. viciae (Rlv) is a soil bacterium which can form nitrogen-fixing symbiotic relationships with leguminous plants. Numerous rhizobial strains found in soils compete with each other. Competition can occur both during the saprophytic growth phase in the rhizosphere and inside plant tissues, during the symbiotic phase. Competition is important as it may affect the composition of rhizobial populations present in the soil and in the root nodules of plants.     

Phosphate solubilization activity of rhizobia native to Iranian soils

Agricultural soils in Iran are predominantly calcareous with very low plant available phosphorus (P) content. In addition to their beneficial N₂-fixing activity with legumes, rhizobia can improve plant P nutrition by mobilizing inorganic and organic P. Isolates from different cross-inoculation groups of rhizobia, obtained from Iranian soils were tested for their ability to dissolve inorganic and organic phosphate. From a total of 446 rhizobial isolates tested for P solubilization by the formation of visible dissolution halos on agar plates, 198 (44%) and 341(76%) of the isolates, solubilized Ca₃(PO₄)₂ (TCP) and inositol hexaphosphate (IHP), respectively. In the liquid Sperber TCP medium, phosphate-solubilizing bacteria (Bacillus sp. and Pseudomonas fluorescens) used as positive controls released an average of 268.6 mg L⁻¹ of P after 360 h incubation. This amount was significantly (P < 0.05) higher than those observed with all rhizobia tested. The group of Rhizobium leguminosarum bv. viciae mobilized in liquid TCP Sperber medium significantly (P < 0.05) more P (197.1 mg L⁻¹ in 360 h) than other rhizobia tested,. This group also showed the highest dissolution halo on the TCP solid Sperber medium. The release of soluble P was significantly correlated with a drop in the pH of the culture filtrates indicating the importance of acid production in the mobilization process. None of the 70 bradyrhizobial isolates tested was able to solubilize TCP. These results indicate that many rhizobia isolated from soils in Iran are able to mobilize P from organic and inorganic sources and this beneficial effect should be tested with crops grown in Iran.     

Genetic Diversity of a Natural Population of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> bv. <Emphasis Type="Italic">viciae</Emphasis> Nodulating Plants of <Emphasis Type="Italic">Vicia faba</Emphasis> in the Vesuvian Area

A total of 98 rhizobial strains, isolated during the winter of the years 2003 (35 isolates), 2004 (33 isolates), and 2005 (30 isolates) were analyzed to determine the genetic diversity of the natural population nodulating Vicia faba plants and to identify dominant genotypes. All isolates were identified as Rhizobium leguminosarum bv. viciae by biovar-specific polymerase chain reaction amplification of the nodC gene. Intraspecific DNA polymorphism was evaluated through the restriction endonucleases analysis combined with pulsed-field gel electrophoresis. Four genotypes characterized 53% of the isolates, showing a high occurrence; moreover, they were recovered over the 3 years, thus showing a lasting persistence in the soil, which could mean a high degree of saprophytic competitiveness. The richness, diversity, and dominance indexes of genotypes were calculated to monitor the evolution of the rhizobial population during the 3 years. The genetic diversity of the analyzed strains decreased along the 3 years. In fact, the biodiversity index H′ decreased from 2.6 in the first and second year to 1.9 in the third year; probably, as a result of bean monocropping, specific genotypes of Rh. leguminosarum bv. viciae were naturally selected.     

The Diversity of Phaseolus-Nodulating Rhizobial Populations Is Altered by Liming of Acid Soils Planted with Phaseolus vulgaris L. in Brazil

PCR-mediated restriction fragment length polymorphism (RFLP) analysis of the 16S-23S rRNA internally transcribed spacer (ITS) region and the 16S rRNA gene indicated that the rhizobial populations isolated from common bean (Phaseolus vulgaris L.) nodules in the unlimed soil from a series of five lime rates applied 6 years previously to plots of an acidic oxisol had less diversity than those from plots with higher rates of liming. Isolates affiliated with Rhizobium tropici IIB and Rhizobium leguminosarum bv. phaseoli were predominant independent of lime application. An index of richness based on the number of ITS groups increased from 2.2 to 5.7 along the soil liming gradient, and the richness index based on “species” types determined by RFLP analysis of the 16S rRNA gene varied from 0.5 to 1.4. The Shannon index of diversity, based on the number of ITS groups, increased from 1.8 in unlimed soil to 2.8 in limed soil, and, based on RFLP analysis of the 16S rRNA gene, ranged from 0.9 to 1.4. In the limed soil, the subpopulation of R. tropici IIB pattern types contained the largest number of ITS groups. In contrast, there were more R. leguminosarum bv. phaseoli types in the unlimed soil with the lowest pH than in soils with the highest pH. The number of ITS (“strain”) groups within R. leguminosarum bv. phaseoli did not change with increased abundance of rhizobia in the soil, while with R. tropici IIB, the number of strain groups increased significantly. Some cultural and biochemical characteristics of Phaseolus-nodulating isolates were significantly related to changes in soil properties caused by liming, largely due to changes in the predominance of the rhizobial species groups.     

Genotypic and symbiotic diversity of native rhizobia nodulating red pea (Lathyrus cicera L.) in Tunisia

Nodulation and genetic diversity of native rhizobia nodulating Lathyrus cicera plants grown in 24 cultivated and marginal soils collected from northern and central Tunisia were studied. L. cicera plants were nodulated and showed the presence of native rhizobia in 21 soils. A total of 196 bacterial strains were selected and three different ribotypes were revealed after PCR-RFLP analysis. The sequence analysis of the rrs and two housekeeping genes (recA and thrC) from 36 representative isolates identified Rhizobium laguerreae as the dominant (53%) rhizobia nodulating L. cicera. To the best of our knowledge, this is the first time that this species has been reported among wild populations of the rhizobia-nodulating Lathyrus genus. Twenty-five percent of the isolates were identified as R. leguminosarum and isolates LS11.5, LS11.7 and LS8.8 clustered with Ensifer meliloti. Interestingly, five isolates (LS20.3, LS18.3, LS19.10, LS1.2 and LS21.20) were segregated from R. laguerreae and clustered as a separate clade. These isolates possibly belong to new species. According to nodC and nodA phylogeny, strains of R. laguerreae and R. leguminosarum harbored the symbiotic genes of symbiovar viciae and clustered in three different clades showing heterogeneity within the symbiovar. Strains of E. meliloti harbored symbiotic genes of Clade V and induced inefficient nodules.     

Growth of Indigenous Rhizobium leguminosarum and Rhizobium meliloti in Soils Amended with Organic Nutrients

The ability of indigenous Rhizobium leguminosarum and Rhizobium meliloti to use organic nutrients as growth substrates in soil was assessed by indirect bacteriophage analysis. A total of 17 organic compounds, including 9 carbohydrates, 3 organic acids, and 5 amino acids, were tested (1,000 μg g⁻¹) in three soils with different cropping histories. Four additional soils were screened with a glucose amendment. Nutrient amendments stimulated growth of indigenous rhizobia, allowing subsequent replication of indigenous bacteriophages. Phage populations were enumerated by plating soil extracts on 19 R. leguminosarum and 9 R. meliloti indicator strains, including root nodule isolates from the soils assayed. On the basis of indirect phage analysis, all soils contained native rhizobia similar to one or more of the indicator strains, although not all indicator strains were detected in soil. All organic compounds stimulated growth of indigenous rhizobia, but the growth response varied for each rhizobial strain depending on the nutrient, the nutrient concentration, and the soil. Indigenous rhizobia readily utilized most organic compounds except phenylalanine, glycine, and aspartic acid. The ability of indigenous rhizobia to utilize a wide range of organic compounds as growth substrates in situ indicates their ability to successfully compete with other soil bacteria for nutrients in these soils.     

Compatibility of Rhizobial Genotypes within Natural Populations of Rhizobium leguminosarum Biovar viciae for Nodulation of Host Legumes

Populations of Rhizobium leguminosarum biovar viciae were sampled from two bulk soils, rhizosphere, and nodules of host legumes, fava bean (Vicia faba) and pea (Pisum sativum) grown in the same soils. Additional populations nodulating peas, fava beans, and vetches (Vicia sativa) grown in other soils and fava bean-nodulating strains from various geographic sites were also analyzed. The rhizobia were characterized by repetitive extragenomic palindromic-PCR fingerprinting and/or PCR-restriction fragment length polymorphism (RFLP) of 16S-23S ribosomal DNA intergenic spacers as markers of the genomic background and PCR-RFLP of a nodulation gene region, nodD, as a marker of the symbiotic component of the genome. Pairwise comparisons showed differences among the genetic structures of the bulk soil, rhizosphere, and nodule populations and in the degree of host specificity within the Vicieae cross-inoculation group. With fava bean, the symbiotic genotype appeared to be the preponderant determinant of the success in nodule occupancy of rhizobial genotypes independently of the associated genomic background, the plant genotype, and the soil sampled. The interaction between one particular rhizobial symbiotic genotype and fava bean seems to be highly specific for nodulation and linked to the efficiency of nitrogen fixation. By contrast with bulk soil and fava bean-nodulating populations, the analysis of pea-nodulating populations showed preferential associations between genomic backgrounds and symbiotic genotypes. Both components of the rhizobial genome may influence competitiveness for nodulation of pea, and rhizosphere colonization may be a decisive step in competition for nodule occupancy.     

Divergent Evolution of Symbiotic Bacteria: Rhizobia of the Relic Legume <Emphasis Type="Italic">Vavilovia formosa</Emphasis> Form an Isolated Group within <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> bv. <Emphasis Type="Italic">viciae</Emphasis>

Comparative sequence analysis of symbiotic genes (nodA, nodC, nodD, nifH), which are elements of accessory component of the rhizobial genome, demonstrated that the strains of Rhizobium leguminosarum bv. viciae, isolated from the nodules of a relic legume, Vavilovia formosa, the closest relative of hypothetical common ancestor of the tribe Fabeae, represented a group separated from the strains of R. leguminosarum bv. viciae, isolated from other representatives of this tribe (Vicia, Lathyrus, Pisum, Lens). No isolation was observed relative to the genes representing the core component of the rhizobial genome (16S rDNA, ITS, glnII) or relative to host specificity of the rhizobia. The data obtained suggest that sequence divergence of symbiotic genes marks the initial stage of sympatric speciation, which can be classified as the isolation of the relic “vaviloviae” symbiotype, a possible evolutionary precursor of the “viciae” biotype.     

Activation of flavonoid biosynthesis in roots of Vicia sativa subsp. nigra plants by inoculation with Rhizobium leguminosarum biovar viciae

Infective (nodulating) Rhizobium leguminosarum biovar viciae (R.l. viciae) bacteria release Nod factors which stimulate the release of nodulation gene-inducing flavanones and chalcones from roots of the host plant Vicia sativa subsp. nigra (K. Recourt et al., Plant Mol Biol 16: 841–852; H.P. Spaink et al., Nature 354: 125–130). The hypothesis that this release results from increased synthesis of flavonoids was tested by studying the effect of inoculation of V. sativa with infective and uninfective R.l. viciae bacteria on (i) activity of L-phenylalanine ammonia-lyase, (ii) level of chalcone synthase mRNA, and (iii) activity of (eriodictyol) methyltransferase in roots. Consistent with the hypothesis, each of these parameters was found to increase 1.5 to 2-fold upon inoculation with infective R.l. viciae bacteria relative to the situation for uninoculated roots and for roots inoculated with uninfective rhizobia.     

Origins of cheating and loss of symbiosis in wild Bradyrhizobium

Rhizobial bacteria nodulate legume roots and fix nitrogen in exchange for photosynthates. These symbionts are infectiously acquired from the environment and in such cases selection models predict evolutionary spread of uncooperative mutants. Uncooperative rhizobia – including nonfixing and non‐nodulating strains – appear common in agriculture, yet their population biology and origins remain unknown in natural soils. Here, a phylogenetically broad sample of 62 wild‐collected rhizobial isolates was experimentally inoculated onto Lotus strigosus to assess their nodulation ability and effects on host growth. A cheater strain was discovered that proliferated in host tissue while offering no benefit; its fitness was superior to that of beneficial strains. Phylogenetic reconstruction of Bradyrhizobium rDNA and transmissible symbiosis‐island loci suggest that the cheater evolved via symbiotic gene transfer. Many strains were also identified that failed to nodulate L. strigosus, and it appears that nodulation ability on this host has been recurrently lost in the symbiont population. This is the first study to reveal the adaptive nature of rhizobial cheating and to trace the evolutionary origins of uncooperative rhizobial mutants.     

Genetic diversity of Vicia faba L. and Pisum sativum L. nodulating rhizobia in the central Black Sea region of Turkey

In this study, we obtained a total of 60 rhizobial isolates from root nodules of Vicia faba L. (n = 30) and Pisum sativum L. (n = 30) grown in the Central Black Sea region of Turkey. The 16S rDNA PCR-RFLP analysis with enzymes CfoI, HinfI, NdeII and MspI revealed a single pattern. Moreover, nucleotide sequence phylogenies based on both the 16S rDNA and recA suggested that these isolates belonged to Rhizobium leguminosarum. Phylogenetic analysis showed that some of our V. faba L.-originated isolates were closely related, indicating molecular evidence for the selection of some special R. leguminosarum bv. viciae isolates by V. faba L., as suggested in previous studies. Network analysis based on recA sequences revealed a common evolutionary history for Turkish, European, North and South American, and Jordanian R. leguminosarum bv. viciae isolates. We isolated four haplotypes using nodA and nifH nucleotide sequence data, i.e. four types of sym plasmids. Two of these types were common to rhizobial isolates from both V. faba L. and P. sativum L., indicating that nodulation factors may not be the mechanism for selection of the special R. leguminosarum bv. viciae populations by V. faba L.     

Bradyrhizobium sp. nodulating the Mediterranean shrub Spanish broom (Spartium junceum L.)

AIMS: The molecular diversity of 25 strains of rhizobia, isolated in Sicily from root nodules of the Mediterranean shrubby legume Spanish broom (Spartium junceum L.), is presented in relation to the known rhizobial reference strains. METHODS AND RESULTS: Our approach to the study of the S. junceum rhizobial diversity combined the information given by the 16S and the intergenic spacer (IGS) 16S-23S rDNA polymorphic region by obtaining them in a single polymerase chain reaction (PCR) step. The PCR fragment size of the S. junceum isolates was 2400-2500 bp and that of the reference strains varied from 2400 in Bradyrhizobium strains to 2800 in Sinorhizobium strains. Inter- and intrageneric length variability was found among the reference strains. Restriction fragment length polymorphisms (RFLP) analysis allowed us to identify eight genotypes among the S. junceum rhizobia that were clustered into two groups, both related to the Bradyrhizobium lineage. Sequencing of representative strains of the two clusters confirmed these data. The 16S-IGS PCR-RFLP approach, when applied to rhizobial reference strains, allowed very close species (i.e. Rhizobium leguminosarum/R. tropici) to be separated with any of the three enzymes used; however, cluster analysis revealed inconsistencies with the 16S-based phylogenesis of rhizobia. CONCLUSIONS: Rhizobia nodulating S. junceum in the Mediterranean region belong to the Bradyrhizobium lineage. Our results confirm the resolution power of the 16S-23S rDNA in distinguishing among rhizobia genera and species, as well as the usefulness of the PCR-RFLP method applied to the entire 16S-IGS region for a rapid tracking of the known relatives of new isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: The present paper is, to our knowledge, the first report on rhizobia nodulating a Mediterranean wild woody legume.     

Characterization of Rhizobial Isolates of Phaseolus vulgaris by Staircase Electrophoresis of Low-Molecular-Weight RNA

Low-molecular-weight (LMW) RNA molecules were analyzed to characterize rhizobial isolates that nodulate the common bean growing in Spain. Since LMW RNA profiles, determined by staircase electrophoresis, varied across the rhizobial species nodulating beans, we demonstrated that bean isolates recovered from Spanish soils presumptively could be characterized as Rhizobium etli, Rhizobium gallicum, Rhizobium giardinii, Rhizobium leguminosarum bv. viciae and bv. trifolii, and Sinorhizobium fredii.     

Identification and Assessment of Symbiotic Effectiveness of Phage-Typed <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> Strains on Lentil (<Emphasis Type="Italic">Lens culinaris</Emphasis> Medik) Cultivars

Symbiotic effectiveness of 19 indigenous and two exotic (USDA 2426 and USDA 2431) strains of lentil Rhizobium belonging to different phage-sensitive and phage-resistant groups was compared under axenic condition. Four strains (USDA 2431, BHULR 104, BHULR 113, and BHULR 115) sensitive to different phages were found significantly superior over others in terms of nodule number, acetylene reduction activity, and total dry weight per plant. Inoculation response of these strains was then evaluated on six lentil cultivars under field condition. A significant symbiotic interaction between rhizobial strains and lentil cultivars was observed. Grain yield enhancement was noticed by the compatible interaction of lentil cultivars HUL-57, L-4147, K-75, and PL-4/DPL-15/DPL-62 with rhizobial strains USDA 2431, BHULR 104, BHULR 113, and BHULR 115, respectively. The authentication of rhizobial strains was accomplished through 16S rDNA sequence analysis. All rhizobial strains had close matching with R. leguminosarum bv. viciae strains. The results have shown that phages can trustfully help selecting out the symbiotically efficient most rhizobial strains for advantageous use with lentil cultivars, in order to strengthen the BNF-based future lentil breeding programs.     

Rhizobium sophorae is the dominant rhizobial symbiont of Vicia faba L. In North China

Faba bean (Vicia faba L.) is a major introduced grain-legume crop cultivated in China. In this study, rhizobia that nodulated faba bean grown in soils from three sites in North China (Hebei Province) were isolated and characterized. Firstly, isolates were categorized into genotypes by ribosomal IGS PCR-RFLP analysis, then representatives of the different IGS genotypes were further identified by phylogenetic analyses of 16S rRNA, housekeeping (atpD, recA) and nodulation (nodC) gene sequences. Rhizobial distribution based on the IGS genotype was related to the different soil physicochemical features by redundancy analysis. IGS typing and phylogenetic analyses of 16S rRNA and concatenated housekeeping gene sequences affiliated the 103 rhizobial strains isolated into four Rhizobium species/genospecies. A total of 69 strains of 3 IGS types were assigned to R. sophorae, 20 isolates of 5 IGS types to R. changzhiense and 9 isolates of 3 IGS types to R. indicum. The representative strain of the five remaining isolates (1 IGS type) was clearly separated from all Rhizobium type strains and was most closely related to defined genospecies according to the recently described R. leguminosarum species complex. Rhizobium sophorae strains (67% of total isolates) were common in all sites and shared an identical nodC sequence typical of faba bean symbionts belonging to symbiovar viciae. In this first study of rhizobia nodulating faba bean in Hebei Province, China, R. sophorae was found to be the dominant symbiont in contrast to other countries.     

Major flavonoids in uninoculated and inoculated roots of Vicia sativa subsp. nigra are four conjugates of the nodulation gene-inhibitor kaempferol

Inoculation of Vicia sativa subsp. nigra (V. sativa) roots with Rhizobium leguminosarum biovar. viciae (R.l. viciae) bacteria substantially increases the ability of V. sativa to induce rhizobial nodulation (nod) genes. This increase is caused by the additional release of flavanones and chalcones which all induce the nod genes of R.l. viciae (K. Recourt et al., Plant Mol Biol 16: 841–852). In this paper, we describe the analyses of the flavonoids present in roots of V. sativa. Independent of inoculation with R.l. viciae, these roots contain four 3-O-glycosides of the flavonol kaempferol. These flavonoids appeared not capable of inducing the nod genes of R.l. viciae but instead are moderately active in inhibiting the activated state of those nod genes. Roots of 7-day-old V. sativa seedlings did not show any kaempferol-glycosidase activity consistent with the observation that kaempferol is not released upon inoculation with R.l. viciae. It is therefore most likely that inoculation with infective (nodulating) R.l. viciae bacteria results in de novo flavonoid biosynthesis and not in liberation of flavonoids from a pre-existing pool.     

Genotypic and phenotypic diversity of rhizobia isolated from Lathyrus japonicus indigenous to Japan

Sixty-one rhizobial strains from Lathyrus japonicus nodules growing on the seashore in Japan were characterized and compared to two strains from Canada. The PCR-based method was used to identify test strains with novel taxonomic markers that were designed to discriminate between all known Lathyrus rhizobia. Three genomic groups (I, II, and III) were finally identified using RAPD, RFLP, and phylogenetic analyses. Strains in genomic group I (related to Rhizobium leguminosarum) were divided into two subgroups (Ia and Ib) and subgroup Ia was related to biovar viciae. Strains in subgroup Ib, which were all isolated from Japanese sea pea, belonged to a distinct group from other rhizobial groups in the recA phylogeny and PCR-based grouping, and were more tolerant to salt than the isolate from an inland legume. Test strains in genomic groups II and III belonged to a single clade with the reference strains of R. pisi, R. etli, and R. phaseoli in the 16S rRNA phylogeny. The PCR-based method and phylogenetic analysis of recA revealed that genomic group II was related to R. pisi. The analyses also showed that genomic group III harbored a mixed chromosomal sequence of different genomic groups, suggesting a recent horizontal gene transfer between diverse rhizobia. Although two Canadian strains belonged to subgroup Ia, molecular and physiological analyses showed the divergence between Canadian and Japanese strains. Phylogenetic analysis of nod genes divided the rhizobial strains into several groups that reflected the host range of rhizobia. Symbiosis between dispersing legumes and rhizobia at seashore is discussed.